Yeast organisms produce a number of proteins that have a function outside the cell. Such proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction (translocation) of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-peptide, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi, the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem. 56:829-852).
Insulin is a polypeptide hormone secreted by xcex2-cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge.
The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids in the configuration: prepeptide - B - Arg Arg - C - Lys Arg -A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains.
Three major methods have been used for the production of human insulin in microorganisms. Two involve Escherichia coli, with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich and Gross, eds.), Pierce Chemical Co., Rockford, Ill. pp 729-739), or use a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a limited number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae, vide U.S. Pat. No. 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.
Circular Dichroism (CD) is used to determine protein stability and relative stabilities of molecules. CD observed below 240 nm is due to the peptide amide chromophore and may be used to estimate protein secondary structure (Johnson (1988) Ann. Rev. Biophys.Chem. 17:145-166). The spectrum of insulin is characterized by minima at 220 and 209 nm, a negative to positive crossover near 203 nm, and a maximum at 195 nm. Upon denaturation, the negative CD in the 240-218-nm range gradually diminishes, consistent with the loss of ordered secondary structure that accompanies protein unfolding. Consequently, the folding stability of an insulin precursor may be quantitated by measuring the loss of secondary structure as a function of added denaturant, e.g., guanidinium hydrochloride (GuHCl) (see e.g., Pace (1975) CRC Crit. Rev. Biochem. 3:1-43).
The present invention features novel connecting peptides (C-peptides) which confer an increased production yield and/or increased stability in insulin precursor molecules and insulin precursor analog molecules when expressed in a transformed microorganism, in particular yeast. Such insulin precursors or insulin precursor analogs can then be converted into insulin or an insulin analog by one or more suitable, well known conversion steps.
The connecting peptides of the present invention contain at least one aromatic amino acid residue Phe, Trp, or Tyr and will generally be shorter than the natural human C peptide which, including the flanking dibasic cleavage sites, consists of 35 amino acids. Thus the novel connecting peptides will in general not be of more than 15 amino acid residues in length and preferably not more than 9 amino acid residues. Typically the novel connecting peptides will be of up to 7 or up to 5 amino acid residues and preferably not more than 4 amino acid residues.
As in the natural human insulin molecule, the connecting peptide will contain a cleavage site at its C and N termini enabling in vitro cleavage of the connecting peptide from the A and B chains. Such cleavage sites may be any convenient cleavage sites known in the art, e.g. a Met cleavable by cyanogen bromide; a single basic amino acid residue or a pair of basic amino acid residues (Lys or Arg) cleavable by trypsin or trypsin like proteases; Acromobactor lyticus protease or by a carboxypeptidase protease. The cleavage site enabling cleavage of the connecting peptide from the A-chain is preferably a single basic amino acid residue Lys or Arg, preferably Lys.
Alternatively, cleavage of the connecting peptide from the B chain may be enabled by cleavage at the natural LysB29 amino acid residue in the B chain giving rise to a desB30 insulin precursor or desB30 insulin precursor analog. The desired B30 amino acid residue may then be added by well known in vitro, enzymatic procedures.
In one embodiment the connecting peptide will not contain two adjacent basic amino acid residues (Lys, Arg). In this embodiment, cleavage from the A-chain may be accomplished at a single Lys or Arg located at the N-terminal end of the A-chain and the natural Lys in position B29 in the B-chain.
The connecting peptide may comprise more than one aromatic amino acid residue but preferably not more than 5. The aromatic amino acid residues may be the same or different. The connecting peptide will preferably not comprise more than 3 aromatic amino acid residues and most preferred it will only comprise a single aromatic amino acid residue.
In one embodiment of the present invention one of the aromatic amino acid residues in the connecting peptide is immediately N-terminal to the cleavage site adjacent to the A chain. Furthermore, one of the aromatic amino acid residues will preferably be positioned less than 5 xc3x85 away from at least one of the residues in positions B11, B12 or B26 in the B chain. In one embodiment, the aromatic amino acid immediately N-terminal to the cleavage site adjacent to the A chain is less than 5 xc3x85 away from at least one of the residues in positions B11, B12 or B26 in the B chain.
The insulin precursors or insulin precursor analogs are characterized by having a high folding stability in solution. The precursors according to the present invention will have an increased Cmid stability compared to insulin or insulin analogs, which do not comprise an aromatic amino acid residue in the connecting peptide. The Cmid stability is thus higher than about 5.5 M GuHCl, typically higher than about 6.0 M GuHCl and more typically higher than about 6.5 M GuHCl.
Accordingly, in one aspect the present invention relates to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains and comprising at least one aromatic amino acid residue and a cleavage site enabling cleavage of the peptide bond between the A-chain and the connecting peptide, wherein one aromatic amino acid residue is immediately N-terminal to said cleavage site.
In another aspect the present invention relates to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains and consisting of up to 9 amino acid residues of which at least one is an aromatic amino acid residue.
In still a further aspect the present invention relates to an insulin precursor or an insulin precursor analog comprising a connecting peptide (C-peptide) being cleavable from the A and B chains, wherein the connecting peptide contains one aromatic amino acid residue which is less than 5 xc3x85 away from at least one of the residues in positions B11, B12 or B26 in the B chain.
In still a further aspect the present invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) comprising at least one aromatic amino acid residue and being cleavable from the A and B chains Said insulin precursors or insulin precursor analogs having an increased Cmid stability relative to insulin precursor or insulin precursor analogs which do not comprise an aromatic amino acid residue a the connecting peptide.
The increased activity is determined by a variety of methods known to one of skill in the art, and described below. In one embodiment, increased stability is measured by CD determination of the concentration of guanidine hydrochloride (GuHCl) needed to achieve half-maximum unfolding of an insulin precursor molecule (Cmid).
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula:
B(1-27)-X2-X3-X1-Y-A(1-21)
wherein
X1 is a peptide sequence of 1-15 amino acid residues comprising one aromatic amino acid residue immediately N-terminal to Y,
X2 is one of Pro, Asp, Lys, or Ile at position 28 of the B chain,
X3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and
Y is Lys or Arg.
In one embodiment, the total number of amino acid residues in X1 will be from 1-10, 1-9, 1-8, 1-7, 1-6, 1-5 or 1-4 amino acid residues in length. In another specific embodiment X1 is 1-3 amino acid residues and preferably 1-2 amino acid residues. The amino acid residues in X1 can be any codable amino acid residue and may be the same or different with the only proviso that one is an aromatic amino acid residue immediately N-terminal to Y.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula:
B(1-27)-X2-X3-X1-Y-A(1-21)
wherein
X1 is a peptide sequence of 1-15 amino acid residues of which one is an aromatic amino acid residue which is less than 5 xc3x85 away from at least one of the amino acid residues in position B11, B12 or B26 in the B chain,
X2 is one of Pro, Asp, Lys, or lie at position 28 of the B chain,
X3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and
Y is Lys or Arg.
In one embodiment the number of amino acid residues in X1 is 1-9, 1-5 or 1-4. In another embodiment the number of amino acid residues is 1-3 or 1-2.
In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula:
B(1-27)-X2-X3-X1-Y-A(1-21)
wherein
X1 is a peptide sequence of 1-8 amino acid residues of which at least one is an aromatic amino acid residue,
X2 is one of Pro, Asp, Lys, or lie at position 28 of the B chain,
X3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and
Y is Lys or Arg.
The total number of amino acid residues in X1 will be from 1-7, 1-6, 1-5 or 1-4 amino acid residues. In a more specific embodiment X1 is 1-3 amino acid residues and preferably 1-2 amino acid residues. The amino acid residues in X1 can be any codable amino acid residue and may be the same or different with the only proviso that at least one amino acid residue in X1 is an aromatic amino acid residue.
In the above formulas X1 may comprise up to 5 aromatic amino acid residues which may be the same or different. In a specific embodiment, X1 comprises up to 3 aromatic amino acid residues which may be the same or different and X1 will preferably contain only one aromatic amino acid residue. The aromatic amino acid residues are Trp, Phe or Tyr, preferably Phe or Trp.
In one embodiment, X2 is Asp and X3 is Lys. This embodiment encompasses the insulin precursor analogs containing an Asp in position B28 of the B chain (termed hereinafter xe2x80x9cAspB28IPxe2x80x9d). In another embodiment X2 is Lys and X3 is Pro. In a further embodiment the sequence X1- Y is selected from the group of:
(a) Met-Trp-Lys; (b) Ala-Trp-Lys; (c) Val-Trp-Lys; (d) Ile-Trp-Lys; (e) Leu-Trp-Lys; (f) Glu-Glu-Phe-Lys (SEQ ID NO:15); (g) Glu-Phe-Lys; (h) Glu-Trp-Lys; (i) Ser-Trp-Lys; (j) Thr-Trp-Lys; (k) Arg-Trp-Lys; (l) Glu-Met-Trp-Lys (SEQ ID NO:1); (m) Gln-Met-Trp-Lys (SEQ ID NO:2); and (n) Asp-Trp-Lys.
In another embodiment X2 is Pro, X3 is Lys and X1 is 1-2 amino acid residues of which one is Trp or Phe.
In another embodiment X2 is Lys, X3 is Pro-Thr and X1 consists of up to 15 amino acid residues of which one is Trp, Tyr or Phe. In this embodiment X1 will contain a cleavage site at the C-terminal end, e.g a mono basic or dibasic (Lys, Arg) cleavage site.
The present invention is also related to polynucleotide sequences which code for the claimed insulin precursors or insulin precursor analogs. In a further aspect the present invention is related to vectors containing such polynucleotide sequences and host cell containing such polynucleotide sequences or vectors.
In another aspect, the invention relates to a process for producing the insulin precursors or insulin precursor analogs in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding the insulin precursors or insulin precursor analogs of the invention under suitable conditions for expression of said precursor or precursor analog; and (ii) isolating the precursor or precursor analog from the culture medium.
In still a further aspect, the invention relates to a process for producing insulin or insulin analogs in a host cell said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or insulin precursor analogs of the invention; (ii) isolating the precursor or precursor analog from the culture medium and (iii) converting the precursor or precursor analog into insulin or an insulin analog by in vitro enzymatic conversion.
In one embodiment of the present invention the host cell is a yeast host cell and in a further embodiment the yeast host cell is selected from the genus Saccharomyces. In a further embodiment the yeast host cell is selected from the species Saccharomyces cerevisiae.